The group A streptococcus (Streptococcus pyogenes, GAS) is a bacterial pathogen of significant medical importance that has evolved specific mechanisms to express an appropriate set of virulence attributes upon encountering a particular host tissue. Mga is a DNA-binding protein of GAS that regulates the transcription of several key virulence genes encoding products essential for colonization and immune evasion of GAS in the host in response to the growth phase and other environmental conditions. Furthermore, orthologues of Mga involved in virulence gene regulation are being found in many other pathogenic streptococci. Thus, Mga provides an excellent system to study global regulatory networks involved in GAS pathogenesis as well as a paradigm for a new family of virulence regulators in Gram-positive pathogens. The overall objectives of this renewal application are (a) to continue a structure/function analysis of Mga and its promoter to better understand the mechanisms by which this key GAS virulence regulator contributes to streptococcal disease, and (b) improve our general understanding of global regulatory pathways that interact to broadly control virulence in these and other important pathogens. The specific aims of this project are (1) To characterize the predicted functional domains within Mga for their role in signal transduction and Mga-dependent transcriptional regulation (2) To examine the mechanisms of Mga-dependent auto-activation and Mga- independent regulation at the mga promoter (Pmga) (3) To establish the role for AmrA and potentially other upstream factors in the growth phase control of mga and the Mga regulon and (4) To investigate how growth phase regulation of the Mga regulon contributes to virulence during GAS infection in mice. A thorough understanding of Mga and its regulation described here will contribute valuable new insights into global virulence regulation not only in GAS, but in other Gram-positive pathogens as well.